Download Interleukin-27 Acts as Multifunctional Antitumor Agent in Multiple

Published OnlineFirst June 29, 2010; DOI: 10.1158/1078-0432.CCR-10-0173
Clinical
Cancer
Research
Cancer Therapy: Preclinical
Interleukin-27 Acts as Multifunctional Antitumor
Agent in Multiple Myeloma
Claudia Cocco1, Nicola Giuliani3, Emma Di Carlo4, Emanuela Ognio2, Paola Storti3, Manuela Abeltino3,
Carlo Sorrentino4, Maurilio Ponzoni5, Domenico Ribatti6, and Irma Airoldi1
Abstract
Purpose: Multiple myeloma (MM) derives from plasmablast/plasma cells that accumulate in the bone
marrow. Different microenvironmental factors may promote metastatic dissemination especially to the
skeleton, causing bone destruction. The balance between osteoclast and osteoblast activity represents a
critical issue in bone remodeling. Thus, we investigated whether interluekin-27 (IL-27) may function as
an antitumor agent by acting directly on MM cells and/or on osteoclasts/osteoblasts.
Experimental Design: The IL-27 direct antitumor activity on MM cells was investigated in terms of
angiogenesis, proliferation, apoptosis, and chemotaxis. The IL-27 activity on osteoclast/osteoblast differentiation and function was also tested. In vivo studies were done using severe combined immunodeficient/nonobese diabetic mice injected with MM cell lines. Tumors from IL-27– and PBS-treated mice
were analyzed by immunohistochemistry and PCR array.
Results: We showed that IL-27 (a) strongly inhibited tumor growth of primary MM cells and MM cell
lines through inhibition of angiogenesis, (b) inhibited osteoclast differentiation and activity and induced
osteoblast proliferation, and (c) damped in vivo tumorigenicity of human MM cell lines through inhibition of angiogenesis.
Conclusions: These findings show that IL-27 may represent a novel therapeutic agent capable of
inhibiting directly MM cell growth as well as osteoclast differentiation and activity. Clin Cancer Res;
16(16); 4188–97. ©2010 AACR.
Interleukin (IL)-27 is a heterodimeric cytokine belonging to the IL-12 family (1). It is composed of the EBVinduced 3 (EBI3) and p28 subunits that are homologues
to the p40 and p35 chains of IL-12, respectively (2). IL-27
is produced by antigen-presenting cells, which protect
their local microenvironment from host pathogens, and
functions on different cell types expressing the full receptor (R) complex (3–7). The IL-27R contains the unique
receptor subunit WSX-1 also known as IL-27Ra/TCCR,
paired with the gp130 chain. Both chains are necessary
Authors' Affiliations: 1Associazione Italiana per la Ricerca sul Cancro
Laboratory of Immunology and Tumors, Department of Experimental
and Laboratory Medicine, G. Gaslini Institute and 2 Animal Model
Facility, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy;
3 Hematology and BMT Center, Department of Internal Medicine and
Biomedical Science, University of Parma, Parma, Italy; 4Department of
Oncology and Neurosciences, “G. d'Annunzio” University and Ce.S.I.
Aging Research Center, “G. d'Annunzio” University Foundation,
Chieti, Italy; 5Pathology Unit, Myeloma Unit, San Raffaele Scientific
Institute, Milan, Italy; and 6 Department of Human Anatomy and
Histology, University of Bari, Bari, Italy
Corresponding Author: Irma Airoldi, Associazione Italiana per la Ricerca
sul Cancro Laboratory of Immunology and Tumors, Department of
Experimental and Laboratory Medicine, G. Gaslini Institute, Genova,
Italy. Phone: 39-0105636342; Fax: 39-0103779820; E-mail: irmaairoldi@
ospedale-gaslini.ge.it.
doi: 10.1158/1078-0432.CCR-10-0173
©2010 American Association for Cancer Research.
4188
for IL-27 signaling (8, 9). IL-27 is considered a proinflammatory and an anti-inflammatory cytokine (10, 11) that
can inhibit TH1 (12–14), TH17 (15, 16), and TH2
responses (17, 18), and suppress the development of inducible regulatory T cells (19). IL-27, similarly to IL-12,
shows antitumor activity in different tumors through indirect mechanisms such as induction of natural killer and
CTL response or inhibition of angiogenesis primarily due
to induction of CXCL10 and CXCL9 (20–26). Recently, it
has been shown that IL-27 inhibits directly the proliferation of human melanoma cell lines expressing functional
IL-27R (27), but there is no information on the role of
IL-27 in human hematologic malignancies.
Multiple myeloma (MM) is a monoclonal postgerminal center tumor that has phenotypic features of plasmablasts/long-lived plasmacells and usually localizes at
multiple sites in the bone marrow (BM; refs. 28). Pathogenesis of MM is complex and dependent on the interactions between tumor cells and microenvironment in
the BM, the primary site of MM development (28,
29). Different cytokines, chemokines, and proangiogenic
factors released in the tumor microenvironment are
known to promote MM cell growth and metastatic dissemination, especially to the skeleton (29–31). MM
represents the second most common hematologic malignancy worldwide, and its prognosis remains grim in
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Published OnlineFirst June 29, 2010; DOI: 10.1158/1078-0432.CCR-10-0173
IL-27 and Multiple Myeloma
Translational Relevance
Multiple myeloma (MM) represents the second
most common hematologic malignancy worldwide,
and its prognosis remains grim in spite of advanced
therapeutic approaches. Here, we show that human
interluekin-27 (IL-27) directly inhibited MM cell
growth both in vitro and in vivo primarily through the
inhibition of angiogenesis. IL-27 also damped the differentiation and activity of osteoclasts, and stimulated
osteoblast proliferation. Our results open new perspectives for MM therapy because IL-27 may block MM
progression and metastatic bone resorption.
spite of advanced therapeutic approaches (32). Thus,
novel therapeutic strategies are warranted to improve
MM prognosis. With this background, we have asked
whether IL-27 may function as antitumor agent against
human MM.
Materials and Methods
Patients
The study design was approved by the Ethical Committee of the University of Parma, Parma, Italy. Twelve MM
patients were studied. Of them, five were male and seven
were female. Patient age ranged from 49 to 92 years. Nine
patients had stage IIIa, two had stage IIIb, and one had
stage Ia disease, according to the Durie and Salmon staging system (33). The monoclonal serum component was
IgGκ in seven cases, IgGλ in four cases, and IgAκ in one
case. BM infiltration with malignant plasma cells at diagnosis ranged from 30% to 75%. At study, all patients were
nontreated. Aliquots of BM aspirates done for clinical evaluation were obtained after informed consent at diagnosis
in 10 cases and at relapse in the remaining two.
Cell culture, antibodies, reagents, and flow cytometry
The human U266, Karpas 620, RPMI 8226, H-Sultan,
OPM-2, JJN3, XG-1, XG-6, and NCI-H929 MM cell lines
(American Type Culture Collection cell bank) were cultured
in RPMI 1640 with 10% FCS (Seromed-BiochromKG).
Human recombinant (hr) IL-27 (R&D System) was used
at different concentrations following titration experiments (50-100 ng/mL in most cases). Fluorochromeconjugated CD19, CD20, CD38, CD138, anti-human IL6,
anti-human IFN-γ, and anti-human immunoglobulin were
from BD Pharmingen. Anti-Ki67 was from DAKO, Dakocytomation, Glostrup, DK. Anti-human WSX-1 monoclonal
antibody (mAb) was from R&D System. Isotype-matched
antibodies of irrelevant specificity (Caltag) were used as
controls. Cells were scored using a FACSCalibur analyzer
(BD Bioscences), and data were processed using CellQuest
software (BD).
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Cell proliferation and apoptosis assays
The human NCI-H929, U266 MM cell lines and the
osteoblastic Hobit cells (kindly gift by Dr. Riggs, Mayo
Clinic, Rochester, MN; ref. 34) were cultured for 5, 16, 24,
48, and 72 hours with or without 100 ng/mL hrIL-27. MM
cells were then stained with Ki67 mAb and analyzed by flow
cytometry. Apoptosis was assessed using AnnexinV/FITC kit
(Bender MedSystems). Hobit cell proliferation was evaluated by MTT Cell proliferation assay (Cayman Chemical). In
some experiments, BM stromal cells (SC), obtained from
BM mononuclear cells of MM patients as previously described (35), were cultured 48 hours in the presence or absence of MM cells from patients (n = 2; BMSC/MM ratio,
1:5) with or without IL-27 (50 ng/mL). Cells were than
tested for apoptosis by flow cytometry
ELISA
Culture supernatants, collected 48 hours after IL-27
treatment, were tested in triplicate using the human
10 cytokine Bio-Plex Assay (IL-12, IFN-γ, IL-10, IL-6,
IL-8, IL-4, IL-5, IL-1β, and tumor necrosis factor α and
β; Bio-Rad Laboratories, Inc.).
Mice studies
Four- to six-week-old severe combined immunodeficientnonobese diabetic (SCID-NOD) mice (Harlan Laboratories) were housed under specific pathogen-free conditions.
All procedures were done in the respect of the National and
International current regulations (D.l.vo 27/01/1992,
n.116, European Economic Community Council Directive
86/609, OJL 358, Dec. 1, 1987).
Two groups of 16 animals each were injected i.p. with
8 × 10 6 NCI-H929 cells. Two additional groups of
10 mice each were injected s.c. with 8 × 106 U266 cells.
One group of mice for each combination was treated
with three weekly doses of hrIL-27 (1 μg/mouse/dose)
starting from 8 hours after tumor cells injection. The
other group of mice from each combination was injected with PBS (controls) according to the same time
schedule. Twenty-one days after tumor cell inoculation,
mice were sacrificed and tumor mass was measured, as
previously described (23).
Chorioallantoic membrane assay
Chorioallantoic membrane (CAM) assay was done as
reported (36) and was loaded with 1 μL of PBS (negative
control), 1 μL of PBS with 250 ng vascular endothelial
growth factor (VEGF; R&D Systems) as positive control,
1 μL of medium from NCI-H929 cells or purified MM
cells from patients cultured 48 hours with or without
100 ng/mL hrIL27, and 1 μL of medium containing
hrIL-27 (100 ng/mL). All supernatants were tested in triplicate, and means ± SD were calculated. CAM were examined daily until day 12 and photographed in ovo with a
stereomicroscope equipped with a camera and image analyzer system (Olympus Italia). On day 12, the angiogenic
response was evaluated by the image analyzer system as
the number of vessels converging toward the sponges.
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Osteoclastogenesis and osteoclast activity
Osteoclast were generated from peripheral blood (PB)
CD14+ monocytes of MM patients (n = 3) or of healthy
donors (n = 3). Cells were isolated by immunomagnetic
beads (Miltenyi Biotec). Monocytes were cultured 28 days
in α-MEM with 10% fetal bovine serum (Invitrogen) and
then plated in eight-well slides (Nalgene Nunc A/S Roskilde) at the density of 7.5 × 105/cm2 with RANKL (Pierce;
50 ng/mL) and M-CSF (Pierce; 25 ng/mL).
Osteoclast phenotype was assessed after 28 days by cytochemical analysis of tartrate-resistant acid phosphatase
(TRAP) using a commercial kit (Sigma Aldrich). HrIL-27
was added (100 ng/mL) to the above monocyte cultures
at day 3, 7, 14, and 21. TRAP+ multinucleated cells containing more than five nuclei (mature osteoclast) were counted
by optical microscopy. Each sample was cultured in duplicate, and 40 microscopy fields at high magnification (×400)
were evaluated for each well. The results were expressed as
mean number of osteoclast counted per well ± SD.
Pit assay was done to evaluate the osteoclast activity. To
this end, osteoclast generated as above and cultured
28 days with or without hrIL-27 were tested using bone
slices (Pantec s.r.l.), and pits were counted by microscopy.
Images were obtained on a Nikon Eclipse TE 300 microscope at 10X/0.13 using a DS-U1 digital slight and at
4×/0.12 objective lens.
Osteoblast marker evaluation and bone
nodule formation
Confluent osteoblastic cells Hobit were incubated in
D-MEM and 10% fetal bovine serum with or without
hrIL-27 (range, 50-100 ng/mL) for 48 hours. Cell lysates
were prepared for Western blot analysis (37) to check collagen and osteocalcin expression. Polyclonal anti-Collagen
I and osteocalcin antibody were from Santa Cruz Biotechnology. In other experiments, confluent Hobit cells were
cultured with ascorbic acid (50 μg/mL) and Dexametasone
(Sigma Aldrich; 1 × 10−8 mol/L), with β-glycerophosphate
(Sigma Aldrich; 10 mmol/L) with or without hrIL-27 (50100 ng/mL) for 1 to 2 weeks. Cells were fixed with cold
formalin at 4°C, washed thrice with PBS, and stained with
Alkaline Phosphatase semiquantitative histochemical kit
(Sigma Aldrich), or fixed with 1:1 mixture (vol/vol) of
37% formaldehyde and ethanol for 5 minutes, washed
thrice with PBS, and stained for 10 minutes with a solution of 2% of Alizarid Red (Sigma Aldrich) at pH 4.2 for
bone nodule evaluation.
PCR array
RNA was extracted from tumors removed from SCIDNOD mice or from CD138 + MM cells cultured for
36 hours with or without (controls) hrIL-27 using Trizol
from Invitrogen. Contaminant genomic DNA was
removed by Dnase treatment (Qiagen GmbH). RNA was
retrotranscribed by the RT2 First Strand cDNA Synthesis
kit (SABioscience).
Human Angiogenesis RT2 PCR Array and RT2 Real-Time
SyBR Green/ROX PCR Mix were from SABioscience. PCR
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was done on the ABI Prism 7700 Sequence Detector (Applied
Biosystems). Gene expression of IL-27–treated and control
samples were analyzed separately in different PCRArray
plates. For each plate, results were normalized on the median value of a set of housekeeping genes. Then, changes
in gene expression between IL-27–treated and control
samples were calculated using the ΔΔCt formula (38).
Results from three different IL-27–treated and control
samples were pooled and, according to the protocol,
analyzed by the software provided by the manufacturer.
A significant threshold of 4-fold change in gene expression
corresponded to P < 0.001.
Histologic and immunohistochemical analyses
Tissue samples were formalin fixed, paraffin embedded,
sectioned at 4-μm-thick sections, and stained with H&E.
For immunohistochemistry, formalin-fixed, paraffinembedded sections were immunostained with rabbit
anti-human laminin (Biogenex), rabbit anti-human IL-6
(Abcam), or rabbit anti-human VEGF-C (Zymed), mouse
anti-human Ki67 (DAKO), rabbit anti-mouse CD31
(Thermo Scientific), and rat anti-mouse F4/80 (Serotec)
antibodies.
After washing, sections were overlaid with goat antirabbit immunoglobulin conjugated to peroxidase-labeled
dextran (EnVision+Peroxidase, rabbit/mouse; Dakocytomation) for 30 minutes. Unbound immunoglobulin was
removed by washing, and slides were incubated with
avidin-biotin complex/alkaline phosphatase (DAKO) for
30 minutes, then sections were counterstained with H&E.
DNA fragmentation associated with apoptosis was detected in 4-μm tissue sections by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining
with the ApopTag Plus Peroxidase In situ Apoptosis kit
(Millipore) according to the manufacturer's protocol.
Statistical methods
Differences in the number of vessels formed in CAM assay and in Hobit cell proliferation cultured in the presence
or absence of IL-27 were evaluated by Student's t test. Differences in tumor volume were calculated using MannWhitney test comparing two independent samples, with
99% confidence interval. All statistical tests were two tailed.
Results
IL-27R in human primary MM cells
First, we investigated the expression of the gp130 and
WSX-1 chains of IL-27R in primary neoplastic cells isolated from the infiltrated BM of 12 MM patients. Figure 1A
shows that WSX-1 expression was consistently detected in
CD138+ MM cells by flow cytometry. The gp130 chain was
always detected in all samples (data not shown).
MM cells are known to release several proangiogenic
factors (39); thus, we asked whether this feature was affected by IL-27. We incubated CD138+ neoplastic cells from
MM patients (patients #1, #2, #4, and #8) with hrIL-27
or medium for 48 hours and tested the angiogenic activity
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IL-27 and Multiple Myeloma
Fig. 1. A, WSX-1 surface expression in human CD138+ primary MM cells. Open profile, WSX-1 staining; dark profile, isotype-matched mAb staining.
B, angiogenic activity of supernatants from one representative (of four) CD138+ MM sample cultured with or without IL-27. CAM-treated with sponges
loaded with the conditioned medium from the nontreated cells were surrounded by allantoic vessels developing radially toward the implant in a spoked
wheel pattern (left). When supernatant from the same MM sample cultured with hrIL27 was tested, a significant reduction of the angiogenic response was
evident (right). Original magnification, ×50. C, CD138+ primary MM cells were incubated with medium (n = 3) or hrIL-27 (n = 3) and tested for mRNA
expression of 84 angiogenesis-related genes. Pooled results of three independent experiments are shown. Histogram represents fold differences of
individual RNA expression between MM cells cultured with or without hrIL-27.
of culture supernatants by CAM assay. There were no significant differences in the proportions of viable cells (trypan blue staining) between cultures done with or without
hrIL-27. CAM treated with sponges loaded with VEGF
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(positive control) or supernatants from primary MM cells
were surrounded by allantoic vessels developing radially
toward the implant in a “spoked wheel” pattern. In the
representative experiment shown in Fig. 1B (left), the
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mean number of vessels formed by supernatant from MM
cells (Pt #1) was 25 ± 4, whereas that formed by VEGF was
30 ± 5 (data not shown). No vascular reaction was detected around the sponges upon exposure to hrIL-27 diluted in medium at the same final concentration used to treat
tumor cells (mean number of vessels, 7 ± 3 with or without IL-27). When the supernatants from hrIL-27–treated
CD138+ cells from the same MM patient was tested, a significant (P < 0.001) reduction of the angiogenic response
was appreciable (mean number of vessels, 12 ± 3; Fig. 1B,
right). Similar results with the same statistical significance
were obtained when supernatants from the three remaining CD138+ primary MM cell suspensions were tested (patient #2: mean number of vessels formed by nontreated
supernatant was 22 ± 3 and 10 ± 2 by hrIL-27-treated supernatant. Patient #4: mean number of vessels formed by
nontreated supernatant was 28 ± 4 and 12 ± 3 by hrIL27–treated supernatant. Patient #8: mean number of vessels formed by nontreated supernatant was 25 ± 2 and
11 ± 2 by hrIL-27–treated supernatant).
Next, we investigated expression of proangiogenic and
antiangiogenic genes in primary MM cells incubated with
hrIL-27 or medium. Purified CD138+ cells from three different MM patients (patients #1, #4, and #8) were cultured
for 36 hours with or without hrIL-27.
Figure 1C shows the pooled results from the three samples analyzed by PCR array. HrIL-27 treatment significantly downregulated mRNA (P < 0.001) of a wide
panel of proangiogenic genes including AKT, angiopoietin 1, angiopoietin 2, alanyl aminopeptidase, CCL2,
laminin 5, matrix metalloproteinase 9, transforming
growth factor β1 and β2, VEGF-A, VEGF-C, and VEGFD, and upregulated mRNA of the antiangiogenic chemokines CXCL9 and CXCL10. Some proangiogenic genes
including CXCL3 and CXCL5 were also upregulated in
the same cells.
In experiments done in triplicate using three different
primary MM cell suspensions, IL-27 did not attract tumor
cells in chemotaxis assays nor affect proliferation, apoptosis, or cytokine release.
IL-27 inhibits osteoclast formation and stimulates
osteoblast proliferation in vitro
The effect of IL-27 on osteoclast and osteoblast was next
investigated. CD14+ monocytes freshly isolated from PB of
MM patients or healthy donors expressed WSX-1, by flow
cytometry (Fig. 2A, left).
Three different osteoclast preparations were generated
from PB CD14+ monocytes of MM patients or normal donors. Cells were stained for TRAP to identify osteoclast.
The number of TRAP-positive osteoclast was significantly
reduced in cultures from either MM patients (Fig. 2A,
right) or normal individuals (data not shown) exposed
to IL-27 on day 3 (P = 0.0004) or 7 (P = 0.003) compared
with those unexposed to the cytokine. Cultures done by
adding or not hrIL-27 on day 14 and 21 (Fig. 2A, right
and data not shown, respectively) contained similar
proportions of osteoclast, indicating that IL-27 had to be
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present in the first week of culture to inhibit osteoclast
differentiation.
In another set of experiments, PB CD14+ monocytes
from MM patients or normal donors cultured for 28 days
with RANKL and M-CSF with or without hrIL-27 were subjected to the Pit assay, which measures resorbed bone area
(Fig. 2B). An inhibitory effect of hrIL-27 on osteoclast activity was shown for cultures from MM patients (mean ± SD
number of pits IL-27 versus control: 15,680 ± 8,700 versus
26,000 ± 7,560; P = 0.028; Fig. 2B). Similar results were obtained with cultures from healthy donors (data not shown).
To test the potential effect of hrIL-27 on osteoblast differentiation and function, we next analyzed WSX-1 expression on osteoblast progenitors and mature osteoblast.
WSX-1 was expressed by the mature osteoblast cells Hobit
(Fig. 2C, left) but not by bone marrow stromal cells
(BMSC) or osteoblast progenitors (35), by flow cytometry.
As shown in Fig. 2C (right), hrIL-27 significantly stimulated (P = 0.044) Hobit cell proliferation, whereas it did not
affect (a) expression of the osteoblast markers Collagen I
and osteocalcin (Fig. 2D, left), (b) expression of alkaline
phosphatase, and (c) bone nodule formation in vitro
(Fig. 2D, middle and right).
In additional experiments, we investigated the potential
ability of BMSC to affect primary myeloma cell growth in
the presence of IL-27. These experiments showed that
IL-27 did not affect apoptosis of CD138+ MM cells cocultured with BMSC (Fig. 2E). As expected, the presence of
BMSC in coculture significantly (P < 0.05) reduced MM
cell apoptosis (Fig. 2E).
hrIL-27 strongly inhibits tumorigenicity of NCI-H929
and U266 cells in SCID-NOD mice
The human NCI-H929, U266, JJN3, RPMI 8226, H-Sultan,
OPM-2, XG-1, XG-6, and Karpas 620 MM cell lines were
analyzed for gp130 and WSX-1 expression. All MM cell
lines constitutively expressed the gp130 (data not shown)
and the WSX-1 chains (three MM cell lines are shown in
Supplementary Fig. 1). Because the NCI-H929 and the
U266 cell lines show functional similarities to the primary
MM cells (Supplementary Fig. 1), they were selected for
in vivo studies. Mice injected i.p. with NCI-H929 cells
and treated with hrIL-27 developed tumors significantly
smaller (P = 0.0047) than mice inoculated with the same
cells that had received PBS (n = 16 for both groups; hrIL27
treated: median volume, 281.6 mm3; range, 5-784 mm3;
PBS treated: median volume, 2,882 mm 3; range, 3806,305 mm3; Fig. 3A, left). Similarly, mice injected s.c. with
the human U266 cells and treated with hrIL-27 developed
tumors significantly smaller than mice inoculated with the
same cells that received PBS (P < 0.0001; n = 10 for both
groups; hrIL-27 treated: median volume, 944.4 mm 3 ;
range, 861-1,163 mm 3 ; PBS treated: median volume,
1,921.8 mm3; range, 1,360-2,404 mm3; Fig. 3A, right).
The angiogenic phenotype of tumors formed in
hrIL-27 versus PBS-treated animals injected with the
human MM cells was investigated. In four different experiments, two performed with tumors from animals
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IL-27 and Multiple Myeloma
Fig. 2. A, left, WSX-1 surface expression in human CD14+ cells from a MM patient. Dark profile, WSX-1 staining; open profile, isotype-matched mAb
staining. Right, osteoclast generated from mononuclear cells of MM patients (n = 3) were identified as TRAP+ multinucleated cells. Columns, mean number
of TRAP+ counted per well; bars, SD. B, pit assay was assessed using osteoclast samples generated from MM patients without (medium, left) or with
(right) hrIL-27. Images shown are representative of one experiment of the three performed and were obtained on a Nikon Eclipse TE 300 microscope at
10X/0.13 using a DS-U1 digital slight and at 4×/0.12 objective lens (original magnification, ×400). C, left, WSX-1 surface expression in human osteoblastic
Hobit cells. Dark profile, WSX-1 staining; open profile, isotype-matched mAb staining. Right, confluent Hobit cells were cultured with or without hrIL-27,
and cell proliferation was evaluated by MTT proliferation assay. Columns, mean of cells from 10 replicate wells done in duplicate. D, left, Collagen I
and osteocalcin expression in Hobit cells treated with or without hrIL-27. Actin was tested as internal control. Middle and right, alkaline phosphatase
expression (middle) and bone nodule formation (right) were evaluated in Hobit cells treated with or without hrIL-27. E, apoptosis of primary MM cells
cocultured with BMSC in the presence or absence of IL-27. Pooled results from two different experiments are shown. MM cells were analyzed by gating
CD138+ cells. MM, primary MM cells cultured with medium alone; MM+IL-27, primary MM cells cultured in the presence of IL-27; MM+BMSC, primary
MM cells cocultured with medium alone in the presence of BMSC; MM+BMSC+IL-27, primary MM cells cocultured with BMSC in the presence of IL-27.
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7,5
5,0
2,5
Fig. 3. A, volume of tumors grown i.p (NCI-H929 cells, PBS treated n = 16; hrIL-27 treated n = 16) and s.c. (U266 cells, PBS treated n = 10; hrIL-27 treated
n = 10) in PBS- and hrIL-27-treated mice. Boxes, values between the 25th and 75th percentiles; whisker lines, highest and lowest values for each
group. Horizontal lines, median values. B, tumor masses from hrIL-27– or PBS-treated mice were tested for expression of 84 human angiogenesis–related
genes by PCR Array. Histogram represents fold differences of individual mRNA expression between hrIL-27 and PBS-treated mice. Top histogram,
genes similarly regulated in both models; bottom histogram, genes differently regulated in both models.
injected with NCI-H929 cells and two with tumors from
mice inoculated with U266 cells, the expression of a
common set of proangiogenic genes was significantly
(P < 0.001) downregulated by IL-27 treatment
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(ANGPTL3, ANGPTL4, CCL2, CXCL3, CXCL5, CXCL6,
EphB4, FGF1, VEGF-C and VEGF-D, VEGFR2, Flt1,
IL-6, IL-8, and CD61; Fig. 3B, top). Other genes were
differentially regulated in the two in vivo models. Thus,
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IL-27 and Multiple Myeloma
MMP9 expression was downregulated only in NCI-H929
tumors formed in IL-27 versus PBS-treated mice. By contrast, expression of endoglin, ephrin A1, ephrin B2,
epidermal growth factor, IFN-α, insulin-like growth factor I, platelet-derived growth factor-α, CD31, and
plateled factor 4 was downregulated only in tumors
formed by U266 cells in hrIL-27 versus PBS-treated animals (Fig. 3B, bottom).
Histologic and immunohistochemical features of
tumors formed by NCI-H929 and U266 cells in
SCID-NOD mice
PBS-treated mice, inoculated s.c. with the human U266
cells, developed poorly differentiated plasma cell myelomas, rarely showing small necrotic areas, and formed by
nests of atypical cells, with occasional double nuclei and
atypical mitotic figures (Fig. 4, a). These tumors were
supplied by well-developed and robust vascular branches
(Fig. 4, b) and showed a distinct expression of IL-6 (Fig. 4,
c) and VEGF-C (Fig. 4, d). Unlike myelomas developed in
control mice, those in hrIL-27–treated mice steadily
showed frequent and wide ischemic-hemorrhagic necrosis
(Fig. 4, e) associated with fragile and heavily damaged vascular branches (Fig. 4, f), and faint IL-6 (Fig. 4, g) and
VEGF-C (Fig. 4, h) expression. No significant differences
between hrIL-27–treated and control mice were observed
in terms of MM cell apoptosis, proliferation, or number of
murine vessels (data not shown). Tumor infiltration with
host mononuclear or polymorphonuclear cells was undetectable in hrIL-27– and PBS-treated animals (data not
shown). Similar histologic and immunohistochemical features were observed in tumors formed after NCI-H929
inoculation i.p. into SCID-NOD mice (data not shown).
Discussion
MM is one of the most common hematological malignancies that originates from the aberrant transformation
and expansion of normal plasmablasts/plasmacells in
the BM (28). MM is characterized by a strong interaction
with BM microenvironment that supports tumor cell
growth and spreading, and by aggressive bone destruction
(28, 29).
We asked whether IL-27 may function as an antitumor
agent in MM disease. Here, we showed that human primary MM cells expressed functional IL-27R, and IL-27 triggering affected their angiogenic activity, whereas it did not
attract tumor cells in chemotaxis assays nor affected their
apoptosis, proliferation, or cytokine release. IL-27 influenced the expression of a wide panel of angiogenesisrelated genes and inhibited different proangiogenic factors
such as VEGF-D and CCL2, two of the major MM growth
factors (40, 41).
IL-27 has been shown to inhibit mouse osteoclast activity by acting on T lymphocytes (6), which are known to
promote osteoclastogenesis in MM (37). In humans, IL-27
has been recently shown to inhibit directly in vitro osteoclast differentiation from mononuclear cells of normal
www.aacrjournals.org
Fig. 4. A, PBS-treated mice developed a poorly differentiated plasma cell
myeloma (a) formed by nests of atypical cells frequently showing
atypical mitotic figures (arrows). This morphologic feature was associated
with a robust vasculature, as evidenced by laminin immunostaining
(b), and a distinct expression of IL-6 (c) and VEGF-C (d). By contrast,
plasma cell myeloma growing in hrIL-27–treated mice (e) showed wide
ischemic-hemorrhagic necrosis (N) associated with fragile and heavily
damaged vascular branches (f) and a very weak IL-6 (g) and VEGF-C
(h) expression. B, terminal deoxynucleotidyl transferase–mediated dUTP
nick end labeling assay (apoptosis), murine vessel (CD31), and
macrophage infiltration (F4/80) in tumors from PBS-treated (a, c, and e)
and hrIL-27-treated (b, d, and f) animals injected with the human U266
cell line.
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Cocco et al.
individuals (42). Whether or not IL-27 can exert a similar
activity in MM patients was unknown. Here, we showed
that osteoclast progenitors from MM patients and controls
expressed functional IL-27R, and that IL-27 directly dampened osteoclast differentiation and bone lytic activity.
IL-27–driven inhibition of the latter activity is consistent
with impairment of osteoclast differentiation operated
by the cytokine.
The balance between osteoclast and osteoblast activity
plays a key role in bone remodeling because it reflects
the balance between bone destruction and new bone formation (43). In advanced metastatic MM, the aberrant
proliferation and activation of osteoclasts overcomes osteoblast activity so strongly that apoptotic osteoblasts can be
observed in lesion sites (44). In this context, we showed
that IL-27 induced proliferation in Hobit cells representing
an immortalized mature osteoblast cell line. However,
IL-27 did not affect other osteoblast activities such as expression of the osteoblast markers Collagen I and osteocalcin, expression of alkaline phosphatase, and bone
nodule formation. Thus, IL-27 seems to amplify the
mature osteoblastic compartment without affecting its
functionality. Other molecules present in the bone microenvironment would complement IL-27 activity by modulating osteoblast function (40).
In immunodeficient SCID-NOD mice injected with two
different MM cell lines using two different routes of inoculation, hrIL-27 strongly inhibited tumor growth. The key
mechanism involved in such effect was the inhibition of
angiogenesis, which caused ischemic necrosis in the tumors. IL-27 induced the downregulation of different
proangiogenic molecules, including some MM growth factors, such as IL-6, VEGF-D, and CCL2 (40, 41). Although
VEGF-D and CCL2 were downregulated in tumors formed
in IL-27–treated mice and in IL-27 in vitro treated primary
MM cells, IL-6 was not detected in primary MM cells. The
latter finding is consistent with previous demonstration
(45) that IL-6 is a paracrine growth factor produced by
stromal cells in BM microenvironment and not by MM
cells themselves.
The use of SCID-NOD mice allowed us to evaluate the
antitumor activity of human IL-27 on human MM cells
in vivo in the absence of immune responses. Although
human IL-27 is not specie specific (2), the following findings support the conclusion that, in our model, the cytokine
targeted directly human MM cells: (a) the in vivo antitumor
mechanisms operated by IL-27 overlapped with those observed in vitro in MM cells, and (b) tumor infiltration with
host mononuclear or polymorphonuclear cells was virtually undetectable. Moreover, tumors formed in IL-27 versus
PBS-treated SCID-NOD mice injected with MM cell lines
showed no differences in terms of number of murine vessels. The latter finding indicated that IL-27–mediated inhibition of angiogenesis was completely dependent on the
direct effect of the cytokine on human MM cells that
damped the formation of tumor-derived endothelial microvessels (46–48).
Taken together, our findings showed for the first time
that human IL-27 acts as a potent antitumor agent for
MM that may block MM progression and metastatic bone
resorption. Additional antitumor effects may be operated
in vivo by IL-27 through enhancement of CTL, natural killer, and CD8+ T-cell cytotoxicity (1). On this ground, IL-27
seems to represent a novel, promising therapeutic agent
for MM patients, whose prognosis remains grim, based
on its multifunctional activity. An additional argument
in favor of this proposal is the low toxicity shown by
IL-27 in animal models, likely in relation to the low induction of IFN-γ in vivo (21).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
Grants from Associazione Italiana per la Ricerca sul Cancro, Milano,
Italy grant no. 4014 (I. Airoldi) and Fondazione Cassa di Risparmio della
Provincia di Chieti (CariChieti), Italy (E.D. Carlo)
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Received 01/20/2010; revised 04/19/2010; accepted 06/08/2010;
published OnlineFirst 06/29/2010.
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Published OnlineFirst June 29, 2010; DOI: 10.1158/1078-0432.CCR-10-0173
Interleukin-27 Acts as Multifunctional Antitumor Agent in
Multiple Myeloma
Claudia Cocco, Nicola Giuliani, Emma Di Carlo, et al.
Clin Cancer Res 2010;16:4188-4197. Published OnlineFirst June 29, 2010.
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